This is a R21 application in response to RFA-DA-02-008. Injection drug users (IDUs) are the single largest risk group for hepatitis C virus (HCV) infection and the co-infections with human immunodeficiency virus (HIV) and HCV are frequently found in IDUs. These two pathogens are also likely to be responsible for the highest infectious disease morbidity and mortality rates among IDUs. The general aim of this study is to determine the role of opioids (e.g., morphine) in the immunopathogenesis of HCV disease in the presence or absence of HIV infection. We hypothesize that opioids, through their receptors on human hepatocytes and immune cells, modulates HCV infection and replication. We will use both in vitro and in vivo models to directly address the question whether opioids have the ability to enhance HCV infection and replication. We seek to understand how HCV and HIV modify each other's replication in both in vitro and in vivo systems. We propose four specific aims: 1) We will determine whether opioids such as morphine enhances HCV infection of and replication in primary human hepatocytes, hepatoma cell lines (HepG2, Huh-7) and T cell lines (MT-2 and MT-2C); 2) We will determine whether opioids have the ability to induce HCV RNA expression in HCV replicon containing human hepatoma cell lines (Huh.5 and Huh.8). We will also examine whether HCV has ability to infect chronically HIV infected human T and monocytic cell lines (ACH-2, J 1.1 and U 1); 3) We will determine whether the removal of CD8+ T cells from PBMCs, and opioids, when added to CD8+ T cell-depleted PBMC isolated from HCV and/or HIV-infected subjects, induce HCV replication. In addition, we will examine whether HIV induces HCV replication in PBMCs isolated from HCV/HIV coinfected subjects; 4) We will determine plasma and PBMC HCV RNA levels in HCV and/or HIV-infected subjects attending methadone program. We will directly measure the levels of HCV RNA in plasma and PBMC using our newly developed real time RT-PCR assay. The investigation of the impact of opioids on HCV infection and its interaction with HIV will contribute to our basic understanding of host defense processes and the role of drug abuse in HCV and/or HIV infection of human liver and immune cells, ultimately further the design and development of improved treatment for drug-abusing patients infected with HCV and/or HIV.